1. Field of the Invention
The present invention relates to a method of calibrating an enzyme immuno assay system and, in particular, to a method of calibrating an enzyme immuno assay system in which an intensity of a chemical luminescence generated in a photometric cell is detected with two optical detectors having different levels of measurement sensitivity.
2. Description of Related Art
An enzyme immuno assay system measures various substances found in blood, such as carcino embryonic antigen (CEA), ferritin (FER), .alpha.-fetoprotein (AFP), and thyroxine binding globulin (TBG). Measurement is accomplished according to the enzyme immuno method, outlined as follows:
A sample (serum) to be measured is put in an antibody immobilized tube with an antibody immobilized therein, and then a suitable enzyme conjugated antibody reagent is added to carry out an immunological reaction. Subsequently, a substrate solution is added to carry out an enzyme reaction, thereby generating a reaction liquid containing hydrogen peroxide A portion of the resulting reaction liquid containing the hydrogen peroxide is then put in a photometric cell, together with a luminescent reagent (luminol), to measure the substances by detecting an intensity of a chemical luminescence generated in said photometric cell.
A conventional chemical luminescence-detecting apparatus is schematically shown in FIG. 7. The conventional apparatus is comprised of a cylindrical photometric cell 91 made of glass or plastic and fixedly mounted on an integrated spherical cell holder 92. A quantity of chemical luminescence generated within the photometric cell 91 is detected by an optical detector photomultiplier tube 94 through a shutter 93, by the so-called batch-type measuring method. A high-voltage power source 95 and an amplifier 96 are also provided.
According to the conventional chemical luminescence-detecting apparatus shown in FIG. 7, only one photomultiplier tube 94 is provided for the photometric cell 91. In the conventional apparatus, it has been necessary to carry out the measurement by regulating the measuring conditions of the photomultiplier tube 94 by various kinds of devices: for example, a supply voltage from said high-voltage power source 95, a value of feedback resistance in said amplifier 96 and the like. Thus, a range in which said quantity of chemical luminescence can be measured is limited because all measurements are carried out under the same condition.
The chemical luminescence method has been recently used to perform enzyme immuno measurement in an enzyme immuno assay. However, because of the inherent range limitation, it has been very difficult to use the above-described conventional chemical luminescence-detecting apparatus for such purpose. The difficulty arises because a large number of items must be randomly measured in the enzyme immuno measurement, resulting in a very wide range in the quantity of light to be measured. The measurement cannot possibly be carried out by means of a single optical detector like that shown in FIG. 7.
The present inventors have made a separate application for patent for an apparatus for detecting a chemical luminescence in which a chemical luminescence generated in a photometric cell is detected by means of a plurality of optical detectors that are different in sensitivity of measurement.
In the case where said apparatus for detecting a chemical luminescence is calibrated by means of, for example, two optical detectors different in sensitivity of measurement, a calibration curve of each optical detector is obtained but outputs from both optical detectors are different in level, so only a noncontinuous calibration curve can be obtained. However, in order to carry out the conversion into concentration over a wide range, a continuous calibration curve is necessary, and the calibration at discontinuous points is also necessary.